ABSTRACT This PPG has established that dysfunction at multiple stages throughout the lysosomal network (LN), comprised of autophagy and the endosomal-lysosomal (EL) pathway, occurs in Alzheimer?s disease (AD) pathogenesis. A hypothesis throughout the PPG is that increasing the levels of ?-site cleaved carboxyl- terminal fragment of the amyloid-? precursor protein (?CTF) through genetic and environmental AD risk factors, including the ApoE4 allele, cholesterol, and recently identified risk genes found by GWAS, disrupts LN function in early-onset AD and in the more common late-onset AD form. In this project 4 (P4), we focus on autophagy alterations driven by late-onset AD risk factors, investigating particularly the role of the ?CTF and its effects on the LN mediated through rab5 activation. The goals will be attained through a comprehensive transcriptomic approach that evaluates all stages of autophagy in homogeneous vulnerable neuronal populations using RNA-sequencing (RNA-seq) and bioinformatics applied to custom-designed pipelines of autophagy and lysosomal genes. These informative and functionally predictive gene analysis pipelines have been confirmed, and are complemented by immunological and biochemical functional analyses of autophagy, including the use of a novel neuron-specific autophagy reporter-construct mouse. Our experimental design overcomes issues associated with the marked cellular heterogeneity of brain tissue, where vulnerable neurons are intermixed with spared neuronal populations and glia, which we show in our preliminary data to have different autophagy pathway programs. The strong validation of this approach derives from our longstanding investigations of autophagy and lysosomal pathways in AD and supports its innovative application to in vivo studies involving specific vulnerable neuronal populations. In conjunction with P1-P3, we will employ a cohort of models, including a wild-type APP overexpressing mouse, a novel rab5 transgenic mouse that displays endosome morphologic and behavioral abnormalities, and humanized ApoE4 mice, among others that display LN deficits relevant to AD, enabling us to test multiple hypotheses related to the pathogenic effects of ?CTF on the autophagy and EL pathways. In Aim 1 we will evaluate the function of all stages of autophagy in vivo through a novel combination of cell-specific transcriptomics, tracking of fluorescence reporter constructs, and parallel immunochemical functional analyses in vulnerable cholinergic, hippocampal, and cortical neurons. Increasing autophagy turnover and inducing lysosomal biogenesis is an appealing therapeutic approach for improving the efficiency of the LN in vulnerable cells, and modalities known to induce autophagy include calorie restriction (CR). In Aim 2 we will test the hypothesis that the benefits of calorie restriction (CR) and an established CR mimetic, epigallocatechin-3-gallate (EGCG), involve increased autophagy turnover and lysosomal biogenesis in vivo, enhancing clearance of substrates by lysosomes including ?CTF, thus averting pathogenic consequences.